Assignment of Downfield Proton Resonances in Purine Nucleoside Phosphorylase·Immucillin-H Complex by Saturation-Transferred NOEs

Hua Deng, Andrzej Lewandowicz, Sean M. Cahill, Richard H. Furneaux, Peter C. Tyler, Mark E. Girvin, Robert Callender, Vern L. Schramm

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and α-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1′, ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4′ is protonated to the cation. It binds tightly to the PNPs with a Kd value 56 pM for human PNP. Previous NMR studies of PNP-ImmH·PO4 have shown that the N-4′ of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (> 11 ppm) are specific to the PNP·ImmH·PO 4 complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using 15N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only ∼1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP·ImmH· PO4 complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP-ImmH complex. The ∼4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to 0-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.

Original languageEnglish (US)
Pages (from-to)1980-1987
Number of pages8
JournalBiochemistry
Volume43
Issue number7
DOIs
StatePublished - Feb 24 2004

Fingerprint

Purine Nucleosides
Protons
Purine-Nucleoside Phosphorylase
Hydrogen bonds
Hydrogen
Hydrogen Bonding
forodesine
Cations
Protonation
Charge distribution
Binding energy
Nucleosides
Sulfhydryl Compounds

ASJC Scopus subject areas

  • Biochemistry

Cite this

Assignment of Downfield Proton Resonances in Purine Nucleoside Phosphorylase·Immucillin-H Complex by Saturation-Transferred NOEs. / Deng, Hua; Lewandowicz, Andrzej; Cahill, Sean M.; Furneaux, Richard H.; Tyler, Peter C.; Girvin, Mark E.; Callender, Robert; Schramm, Vern L.

In: Biochemistry, Vol. 43, No. 7, 24.02.2004, p. 1980-1987.

Research output: Contribution to journalArticle

Deng, Hua ; Lewandowicz, Andrzej ; Cahill, Sean M. ; Furneaux, Richard H. ; Tyler, Peter C. ; Girvin, Mark E. ; Callender, Robert ; Schramm, Vern L. / Assignment of Downfield Proton Resonances in Purine Nucleoside Phosphorylase·Immucillin-H Complex by Saturation-Transferred NOEs. In: Biochemistry. 2004 ; Vol. 43, No. 7. pp. 1980-1987.
@article{f453ba873e274388a7311c7255329702,
title = "Assignment of Downfield Proton Resonances in Purine Nucleoside Phosphorylase·Immucillin-H Complex by Saturation-Transferred NOEs",
abstract = "Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and α-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1′, ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4′ is protonated to the cation. It binds tightly to the PNPs with a Kd value 56 pM for human PNP. Previous NMR studies of PNP-ImmH·PO4 have shown that the N-4′ of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (> 11 ppm) are specific to the PNP·ImmH·PO 4 complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using 15N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only ∼1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP·ImmH· PO4 complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP-ImmH complex. The ∼4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to 0-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.",
author = "Hua Deng and Andrzej Lewandowicz and Cahill, {Sean M.} and Furneaux, {Richard H.} and Tyler, {Peter C.} and Girvin, {Mark E.} and Robert Callender and Schramm, {Vern L.}",
year = "2004",
month = "2",
day = "24",
doi = "10.1021/bi0358115",
language = "English (US)",
volume = "43",
pages = "1980--1987",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Assignment of Downfield Proton Resonances in Purine Nucleoside Phosphorylase·Immucillin-H Complex by Saturation-Transferred NOEs

AU - Deng, Hua

AU - Lewandowicz, Andrzej

AU - Cahill, Sean M.

AU - Furneaux, Richard H.

AU - Tyler, Peter C.

AU - Girvin, Mark E.

AU - Callender, Robert

AU - Schramm, Vern L.

PY - 2004/2/24

Y1 - 2004/2/24

N2 - Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and α-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1′, ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4′ is protonated to the cation. It binds tightly to the PNPs with a Kd value 56 pM for human PNP. Previous NMR studies of PNP-ImmH·PO4 have shown that the N-4′ of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (> 11 ppm) are specific to the PNP·ImmH·PO 4 complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using 15N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only ∼1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP·ImmH· PO4 complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP-ImmH complex. The ∼4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to 0-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.

AB - Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and α-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1′, ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4′ is protonated to the cation. It binds tightly to the PNPs with a Kd value 56 pM for human PNP. Previous NMR studies of PNP-ImmH·PO4 have shown that the N-4′ of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (> 11 ppm) are specific to the PNP·ImmH·PO 4 complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using 15N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only ∼1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP·ImmH· PO4 complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP-ImmH complex. The ∼4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to 0-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.

UR - http://www.scopus.com/inward/record.url?scp=1242285470&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1242285470&partnerID=8YFLogxK

U2 - 10.1021/bi0358115

DO - 10.1021/bi0358115

M3 - Article

C2 - 14967038

AN - SCOPUS:1242285470

VL - 43

SP - 1980

EP - 1987

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 7

ER -